PROJECT SUMMARY The sphingolipid (SL) ceramide is a potent tumor suppressor that contributes to the promotion of apoptosis and autophagy. Management of these responses in cancer cells is dependent on the dynamic balance between ceramide and its metabolites, some of which can promote cell survival. Maintaining elevated levels of intracellular ceramide is important for supporting its unique anticancer properties. Thus, from a strategic point, controlling the metabolism of ceramide offers new opportunities for regulating cancer growth. The goal of this work is to assess innovative steps to develop ceramide into an effective anticancer agent to treat acute myelogenous leukemia (AML), the most common type of leukemia in adults, and to evaluate the role of SLs in chemotherapy resistance. There is a critical need to develop more effective therapies for AML, especially in relapse patients. Although many hypotheses have been proposed to explain therapeutic relapse, none have led to a complete understanding of the molecular mechanisms of AML resistance, and there have been few treatment advances in 40 years. Our premise is that upregulated glycosylation of ceramide is the major metabolic avenue contributing to ceramide neutralization in cancer cells, and it is also strongly associated with the multidrug- resistant phenotype in cancer. This project will focus on targeting ceramide glycosylation to enhance and propel ceramide-driven AML cell death. Two innovative strategies will be employed to increase intracellular ceramide levels: 1) use of a nanoliposomal, cell-permeable ceramide analog, C6-ceramide (CNL, ceramide nanoliposome), and 2) employment of ceramide ?generators?, drugs such as 4-HPR (fenretinide) or PSC833 (valspodar), that increase intracellular levels of natural long-chain ceramides. These strategies will be evaluated separately, in combination, and in the presence of agents that inhibit ceramide glycosylation. We hypothesize that taking steps to pharmacologically increase and maintain intracellular ceramide levels will enhance the overall anticancer impact of ceramide-centric therapy. Further, given new findings on chemotherapy selection pressure in AML, we hypothesize that aberrant SL metabolism plays a key role in drug resistance and that targeting SL metabolism will potentially circumvent drug resistance. The following three Specific Aims will be pursued to achieve our goal of employing ceramide and SL therapy in AML. 1. Determine the effects of chemotherapy selection pressure on SL metabolism and the drug resistant phenotype in AML. 2. Determine the effects of inhibitors of ceramide glycosylation on cytotoxicity and mechanism of action of CNL and ceramide-generating drugs. 3. Assess the anticancer activity of ceramide-centric therapeutics in AML preclinical models. We predict that using adjuvants to block ceramide glycosylation will enhance ceramide-centric (CNL + ceramide ?generators?) therapy in AML.